In this Merit Award extension application, the overarching goal will continue to be the understanding of fundamental mechanisms involved in acute regulation of insulin secretion by glucose and other metabolic fuels. The specific aims of the project for the next five years are: 1) To test the long-chain acylCoA hypothesis of GSIS, which holds that anintact link betweenglucose andlipidmetabolism is required for glucose sensing. Small interfering RNA (siRNA) technology will be used to modulate expression of key enzymes and mediators of metabolic function that link glucose and lipid metabolism in beta-cells, including citrate lyase, acetyl CoA carboxylase-1 (ACC-1), and the GPR40 receptor. Comprehensive metabolic analysis will be conducted, including glucose usage, glucose oxidation, glucose conversion into lipids, pyruvate cycling by 13C NMR, and metabolic profiling of organic acids, acylcarnitines, amino acids, and free fatty acids by GC/MS and MS/MS; 2) To test a hypothesis concerning the role of pyruvate recycling in regulation of glucose-stimulated insulin secretion. We seek to identify the specific pyruvate cycling pathway(s) that mediates GSIS via comprehensive metabolic analysis in beta-cells subjected to the following molecular manipulations: a) suppression and overexpression of pyruvate carboxylase; b) suppression and overexpression of PDK-1 or PDK-2; c) suppression and overexpression of cytosolic or mitochondrial malic enzymes; d) suppression and overexpression of the dicarboxylate carrier (DIG), that mediates transport of malate from the mitochondria to the cytosol; e) suppression and overexpression of enoyl CoA hydratase (ECH); 3) To investigate two hypothesesabout the potential link between NADPH and regulation of insulin secretion Two hypotheses will be investigated: a) NADPH regulates insulin secretion by binding to the beta-subunit of voltage-gated K (Kv) channels, prolonging the repolarization phase of the plasma membrane current; b) NADPH produced by the pyruvate cycling enzymes ICDc and MEc controls expression of neuroendocrine protein 7B2, a regulator of the proinsulin processing enzyme PC2, thereby affecting proinsulin:insulin ratios; 4) To translate information about new targets for regulation of insulin secretion from the in vitro to the in vivo setting using UTMD technology.